Determination of the sequence of DNA, RNA, and peptide fragments continues to play a significant role in the development of diagnostic medicine, forensics, molecular biology research, and pharmaceutical pharmacogenetics. However, more recently attention has turned from the determination of sequence itself to identification of the function of sequences in biochemical pathways and disease states. Because the genetic influence on most biochemical pathways has been more complex than originally thought, typically involving multiple genes, multiple mutations in genes, and complex interactions, the need to improve the productivity to perform simultaneously multiple assays of DNA sequence has grown.
One way to achieve many parallel simultaneous measurements is to lay out a large number of DNA, RNA, or peptide probe assays onto a microarray which can then be probed simultaneously by complex biological samples. Each individual probe in the array, through hybridization (specific bonding), or not, with, for example, DNA or RNA in an unknown sample, provides information about the presence or absence of sequences in the sample.
These types of assays typically test for the presence of a specific nucleic acid sequence, usually a DNA or RNA sequence, although other specific binding assays are also possible. As is well known in the field, this is accomplished by utilizing oligonucleotides synthesized with specific, predetermined sequences of interest. Typically such specific sequence is based on searches of genome or mutation databases or, for example, homology to a known or putative gene or amino acid sequence, or catalogued mutations of such sequences. The presence or absence of many sequences can then be ascertained simultaneously by hybridization under conditions which allow only perfectly or closely matching sequences to associate.
There are numerous examples of important multiple assays performed by simultaneous microarray analysis. To improve productivity in disease diagnosis, an array can be made in which 500 different probes, each one corresponding to a mutation known to cause, for example, Cystic Fibrosis, are hybridized with a patient's DNA such that, if any of the causative mutations are present, that specific feature of the array representing that mutation becomes fluorescent. In another useful example, DNA sequences corresponding to many genes whose functions are unknown are formed into a microarray. Messenger RNA prepared from both normal and diseased tissue samples can then be compared by measuring differential intensity of probe hybridization on the many different sequences corresponding to many different genes simultaneously. Those genes hybridized differently in the disease tissue compared to the normal tissue can then be implicated in the disease pathway, and assigned a function. Additionally there are numerous examples in molecular biology and pharmaceutical discovery in which the presence or absence of large number DNA sequences need to be analyzed to determine important specifics of a disease state, e.g., resistance to antibiotics, genotype indicative of severity, etc.
Finally, there are many applications in which drug/receptor interactions can be determined by tethering the candidate drugs (such as small organic molecules) or biological receptors to a microarray surface and observing the degree to which the two associate.
Thus, it is often necessary to create a large number of related, but distinct chemical features on a microarray. Synthesis of arrays of bound oligonucleotides or peptides is also generally known in the art. In one approach to parallel synthesis, known as the T-bag method or disk design, an array of individual packets or disks of solid support beads are physically sorted into four (4) amidite subsets for treatment with the selected amidite. After each packet of beads has been treated with the common reagent, the packets must again be manually resorted into the four subsets for the subsequent synthesis cycle. Such sorting and resorting becomes too burdensome and labor intensive for the preparation of large arrays of oligonucleotides.
Another array approach for the synthesis of support-bound oligonucleotides is that of Southern et. al., U.S. Pat. No. 5,436,327, which performs the synthesis in a very narrow gap between two glass plates. Not only is this technique impractical and cumbersome in practice since a plurality of different reagents must be applied accurately to specific sites on the glass surface, but this approach does not allow a continuous synthesis of oligonucleotides. Moreover, since Southern uses standard reagents for phosphoramidite synthesis, the technique needs to be performed in a closed environment to prevent rapid evaporation of the highly volatile solvents (E.g., acetonitrile and dichloromethane, as will be described in greater detail below).
One preferred approach for synthesis of arrays of oligonucleotides on open solid support surfaces is described by Brennan, U.S. Pat. No. 5,474,796, which controls delivery of specific reagents through drop-on-demand inkjet devices. Brennan provides a general method for conducting a large number of chemical reactions on a support surface where very minute volumes of solutions of chemical reactants are added to functionalized binding sites on the support surface by means of a piezo electric pump. The functional binding sites are separated from each other by the use of a non-functionalized coating material with different surface tension.
While Brennan does not specify the nature of the solvents employed with this system in order to make this open surface method applicable to general chemical synthesis, in practice, most chemical reactions are performed in highly volatile, low-boiling solvents such as acetonitrile or dichloromethane. One problem associated with using these conventional solvents in open synthesis arrangements is that when the delivered drops are too small, the solvents tend to evaporate too quickly. This is especially true with the piezo electric delivery pump devices, as applied in Brennan, since the volume of delivered drops are typically between about 20 picoliters to 2 microliters. In this minute size range, the vapor pressure and surface area to total volume ratio of the drops are so high that these standard high yield DNA synthesis solvents evaporate before reacting completely.
The rate of evaporation is a function of the surface area of the drop, and is related to 1/R.sup.2 (where R is the radius of the drop), i.e. the smaller the drop, the faster the rate of evaporation. At 23.degree. C., a 100 micron droplet of acetonitrile (ACN) will evaporate in flight within 1 cm of travel. Once the solvent has evaporated, the amidite coupling reaction essentially ceases in the thick, gummy or crystalline residue.
In conventional solid phase oligonucleotide synthesis on controlled pore glass (CPG), for example, the preferred solvent for the tetrazole activated, dimethoxytrityl protected nucleotide phosphoramidite coupling step is acetonitrile. This solvent has been determined to be far superior to other common solvent types for phoshoramidite coupling, such as tetrahydrofuran, dimethoxyethane and nitromethane. Acetonitrile possesses the ideal combination of acidity, viscosity, dielectric constant solubility and other properties to promote high (&gt;99%) yield coupling. A stepwise coupling yield less than 97% would produce a useless mixture of truncated and/or deleted products.
Acetonitrile (ACN) unfortunately has a rather low boiling point (81.degree. C.), and drops on a support surface evaporate very quickly in an open system. The other above-indicated commonly employed solvents for DNA synthesis (i.e., tetrahydrofuran, dimethoxyethane and nitromethane) also have similar boiling points and rates of evaporation as acetonitrile. Accordingly, these solvents tend to evaporate very quickly in open synthesis systems as well.
One solution would be to deliver a rapid series of droplets of reagent to build up a larger volume for the larger diameter reaction sites. However, this technique becomes ineffective for diameters less than about 500 microns, and diameters greater than 500 microns are far too large in many array approaches Moreover, the rate of evaporation may be slowed by lowering the temperature of the solvent and reaction surface, but the rate of the coupling reaction may be severely reduced. Finally, the rate of evaporation of a drop can also be slowed by increasing the relative humidity or saturation of the head space vapor by ACN. In practice however, stable ACN humidity control is difficult to achieve, and the synthesis device tends to function as a cloud chamber.